FabZ

ABSTRACT

The invention provides fabZ polypeptides and polynucleotides encoding fabZ polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing fabZ polypeptides to screen for antibacterial compounds.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the Fatty acidbiosynthetic pathway family, as well as their variants, hereinafterreferred to as “fabZ,” “fabZ polynucleotide(s),” and “fabZpolypeptide(s)” as the case may be.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studies microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains that are resistantto some or all of the standard antibiotics. This phenomenon has createdan unmet medical need and demand for new anti-microbial agents,vaccines, drug screening methods, and diagnostic tests for thisorganism.

Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the fabZ embodiments of the invention, that have a present benefitof, among other things, being useful to screen compounds forantimicrobial activity. Such factors are also useful to determine theirrole in pathogenesis of infection, dysfunction and disease. There isalso a need for identification and characterization of such factors andtheir antagonists and agonists to find ways to prevent, ameliorate orcorrect such infection, dysfunction and disease.

SUMMARY OF THE INVENTION

The present invention relates to fabZ, in particular fabZ polypeptidesand fabZ polynucleotides, recombinant materials and methods for theirproduction. In another aspect, the invention relates to methods forusing such polypeptides, including treatment of microbial diseases,amongst others. In a further aspect, the invention relates to methodsfor identifying agonists and antagonists using the materials provided bythe invention, and for treating microbial infections and conditionsassociated with such infections with the identified agonist orantagonist compounds. In a still further aspect, the invention relatesto diagnostic assays for detecting diseases associated with microbialinfections and conditions associated with such infections, such asassays for detecting fabZ expression or activity.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to fabZ polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a fabZ of Streptococcuspneumoniae, which is related by amino acid sequence homology to fabZpolypeptide. The invention relates especially to fabZ having thenucleotide and amino acid sequences set out in Table 1 as SEQ ID NO:1and SEQ ID NO:2 respectively. Note that sequences recited in theSequence Listing below as “DNA” represent an exemplification of theinvention, since those of ordinary skill will recognize that suchsequences can be usefully employed in polynucleotides in general,including ribopolynucleotides.

TABLE 1 FabZ Polynucleotide and Polypeptide Sequences (A) Streptococcuspneumoniae fabZ polynucleotide sequence [SEQ ID NO:1].5′-ATGATCGATATTCAAGGAATCAAAGAAGCTCTTCCCCACCGTTATCCTATGCTTCTAGTGGACCGTGTCTTGGAAGTGAGCGAGGATACCATTGTTGCTATCAAAAATGTGACCATCAACGAGCCTTTCTTTAACGGCCACTTTCCTCAATACCCAGTTATGCCAGGTGTTGTGATTATGGAAGCCTTGGCGCAAACTGCCGGTGTGTTGGAGTTATCAAAACCTGAAAATAAAGGAAAACTGGTCTTTTACGCTGGTATGGACAAGGTTAAGTTCAAGAAGCAAGTTGTACCAGGCGACCAATTGGTTATGACAGCGACTTTTGTAAAACGTCGTGGCACCATAGCTGTGGTTGAAGCAAAGGCTGAAGTGGATGGCAAGCTTGCAGCCAGTGGTACCCTTACTTTTGCAATTGGGAAC TAA-3′ (B)Streptococcus pneumoniae fabZ polypeptide sequence deduced from apolynucleotide sequence in this table [SEQ ID NO:2].NH₂-MIDIQGIKEALPHRYPMLLVDRVLEVSEDTIVAIKNVTINEPFFNGHFPQYPVMPGVVIMEALAQTAGVLELSKPENKGKLVFYAGMDKVKFKKQVVPGDQLVMTATFVKRRGTIAVVEAKAEVDGKLAASGTLTFAIGN—COOH

Deposited Materials

A deposit containing a Streptococcus pneumonia 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit. On Apr.17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.The Streptococcus pneumonia strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains a full length fabZ gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain, which polypeptide is containedin the deposited strain. Further provided by the invention are fabZpolynucleotide sequences in the deposited strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare fabZ polypeptide and polynucleotide sequences isolated from thedeposited strain.

Polypeptides

FabZ polypeptide of the invention is substantially phylogeneticallyrelated to other proteins of the fatty acid biosynthetic pathway family.

In one aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as “fabZ” and “fabZpolypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

Among the particularly preferred embodiments of the invention arevariants of fabZ polypeptide encoded by naturally occurring alleles ofthe fabZ gene.

The present invention further provides for an isolated polypeptidewhich: (a) comprises or consists of an amino acid sequence which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, mostpreferably at least 97-99% or exact identity, to that of SEQ ID NO:2over the entire length of SEQ ID NO:2 or a functional fragment thereof,(b) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ IDNO:1; (c) a polypeptide encoded by an isolated polynucleotide comprisingor consisting of a polynucleotide sequence encoding a polypeptide whichhas at least 70% identity, preferably at least 80% identity, morepreferably at least 90% identity, yet more preferably at least 95%identity, even more preferably at least 97-99% or exact identity, to theamino acid sequence of SEQ ID NO:2, over the entire length of SEQ IDNO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity offabZ, and also those which have at least 70% identity to a polypeptideof Table 1 [SEQ ID NO:1] or the relevant portion, preferably at least80% identity to a polypeptide of Table 1 [SEQ ID NO:2 and morepreferably at least 90% identity to a polypeptide of Table 1 [SEQ IDNO:2] and still more preferably at least 95% identity to a polypeptideof Table 1 [SEQ ID NO:2] and also include portions of such polypeptideswith such portion of the polypeptide generally containing at least 30amino acids and more preferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolymer or a homopolyer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStreptococcus pneumoniae, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. As with fabZ polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Streptococcus pneumonia, are also preferred.Further preferred are fragments characterized by structural orfunctional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Also preferred are biologically active fragments that are thosefragments that mediate activities of fabZ, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause Disease in anindividual, particularly a human.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

In addition to the standard single and triple letter representations foramino acids, the term “X” or “Xaa” may also be used in describingcertain polypeptides of the invention. “X” and “Xaa” mean that any ofthe twenty naturally occurring amino acids may appear at such adesignated position in the polypeptide sequence.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodefabZ polypeptides, particularly polynucleotides that encode thepolypeptide herein designated fabZ.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding fabZ polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1] which includes a full lengthgene, or a variant thereof. The Applicants believe that this full lengthgene is essential to the growth and/or survival of an organism thatpossesses it, such as Streptococcus pneumoniae.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding an/or expressing fabZ polypeptides andpolynucleotides, particularly Streptococcus pneumoniae fabZ polypeptidesand polynucleotides, including, for example, unprocessed RNAs, ribozymeRNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments ofthe invention include biologically, diagnostically, prophylactically,clinically or therapeutically useful polynucleotides and polypeptides,and variants thereof, and compositions comprising the same.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a fabZ polypeptidehaving a deduced amino acid sequence of Table 1 [SEQ ID NO:2] andpolynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is afabZ polypeptide from Streptococcus pneumonia comprising or consistingof an amino acid sequence of Table 1 [SEQ ID NO:2], or a variantthereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding fabZ polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Streptococcus pneumoniae 0100993 cellsas starting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as apolynucleotide sequence given in Table 1 [SEQ ID NO:1], typically alibrary of clones of chromosomal DNA of Streptococcus pneumoniae 0100993in E. coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent hybridization conditions. By sequencingthe individual clones thus identified by hybridization with sequencingprimers designed from the original polypeptide or polynucleotidesequence it is then possible to extend the polynucleotide sequence inboth directions to determine a full length gene sequence. Conveniently,such sequencing is performed, for example, using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Direct genomic DNA sequencing may also be performed toobtain a full length gene sequence. Illustrative of the invention, eachpolynucleotide set out in Table 1 [SEQ ID NO:1] was discovered in a DNAlibrary derived from Streptococcus pneumoniae 0100993.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon which begins at nucleotide number 421 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynucleotidesequence which has at least 70% identity, preferably at least 80%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, even more preferably at least 97-99% or exactidentity to SEQ ID NO:1 over the entire length of SEQ ID NO:1; (b) apolynucleotide sequence encoding a polypeptide which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or 100% exact, to the amino acid sequence ofSEQ ID NO:2, over the entire length of SEQ ID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Streptococcuspneumoniae, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof; andisolating a full-length gene and/or genomic clones containing saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86; 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof which may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 421 set forth in SEQ ID NO:1 ofTable 1, both of which encode the fabZ polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3′ end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5′ and nucleic acid residue is at the left,bound to R₁, and its 3′ end nucleic acid residue is at the right, boundto R₃. Any stretch of nucleic acid residues denoted by either R₁ and/orR₂, where m and/or n is greater than 1, may be either a heteropolymer ora homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, which can bea double-stranded polynucleotide wherein the formula shows a firststrand to which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Streptococcus pneumoniae, however, it may preferably be obtainedfrom other organisms of the same taxonomic genus. A polynucleotide ofthe invention may also be obtained, for example, from organisms of thesame taxonomic family or order.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae fabZhaving an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingfabZ variants, that have the amino acid sequence of fabZ polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, modified, deleted and/oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of fabZ polypeptide.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding fabZ polypeptide having an amino acid sequences set out inTable 1 [SEQ ID NO:2], and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding fabZ polypeptide andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to fabZ polynucleotide sequences, such as thosepolynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook et al, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein. Solution Hybridization may also be used with thepolynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said polynucleotide sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding fabZ and to isolate cDNAand genomic clones of other genes that have a high identity,particularly high sequence identity, to the fabZ gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have lee than 30 nucleotide residues or basepairs.

A coding region of a fabZ gene may be isolated by screening using a DNAsequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is the mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to the mature polypeptide (when themature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from the mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived form the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells which are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci, E. coli,streptomyces, cyanobacteria, Bacillus subtilis, and Streptococcuspneumoniae; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of fabZ polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof fabZ polynucleotides and/or polypeptides in a eukaryote, particularlya mammal, and especially a human, will provide a diagnostic method fordiagnosis of disease, staging of disease or response of an infectiousorganism to drugs. Eukaryotes, particularly mammals, and especiallyhumans, particularly those infected or suspected to be infected with anorganism comprising the fabZ gene or protein, may be detected at thenucleic acid or amino acid level by a variety of well known techniquesas well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled fabZ polynucleotide sequences.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA respectively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et al., Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, V1 and S1 protection assay or achemical cleavage method. See, for example, Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingfabZ nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit which comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, which isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The nucleotide sequences of the present invention are also valuable fororganism chromosome identification. The sequence is specificallytargeted to, and can hybridize with, a particular location on anorganism's chromosome, particularly to a Streptococcus pneumoniaechromosome. The mapping ofrelevant sequences to chromosomes according tothe present invention may be an important step in correlating thosesequences with pathogenic potential and/or an ecological niche of anorganism and/or drug resistance of an organism, as well as theessentiality of the gene to the organism. Once a sequence has beenmapped to a precise chromosomal location, the physical position of thesequence on the chromosome can be correlated with genetic map data. Suchdata may be found on-line in a sequence database. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through known genetic methods, for example,through linkage analysis (coinheritance of physically adjacent genes) ormating studies, such as by conjugation.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding fabZ polypeptide can be used to identify andanalyze mutations. These primers may be used for, among other things,amplifying fabZ DNA and/or RNA isolated from a sample derived from anindividual, such as a bodily material. The primers may be used toamplify a polynucleotide isolated from an infected individual, such thatthe polynucleotide may then be subject to various techniques forelucidation of the polynucleotide sequence. In this way, mutations inthe polynucleotide sequence may be detected and used to diagnose and/orprognose the infection or its stage or course, or to serotype and/orclassify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a fabZ polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of fabZ polypeptide compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a fabZpolypeptide, in a sample derived from a host, such as a bodily material,are well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis,antibody sandwich assays, antibody detection and ELISA assays.

Differential Expression

The polynucleotides and polynucleotides of the invention may be used asreagents for differential screening methods. There are many differentialscreening and differential display methods known in the art in which thepolynucleotides and polypeptides of the invention may be used. Forexample, the differential display technique is described by Chuang etal., J. Bacteriol. 175:2026-2036 (1993). This method identifies thosegenes which are expressed in an organism by identifying mRNA presentusing randomly-primed RT-PCR. By comparing pre-infection and postinfection profiles, genes up and down regulated during infection can beidentified and the RT-PCR product sequenced and matched to ORF“unknowns.”

In Vivo Expression Technology (IVET) is described by Camilli et al.,Proc. Nat'l Acad. Sci. USA. 91:2634-2638 (1994). IVET identifies genesup-regulated during infection when compared to laboratory cultivation,implying an important role in infection. ORFs identified by thistechnique are implied to have a significant role in infectionestablishment and/or maintenance. In this technique random chromosomalfragments of target organism are cloned upstream of a promoter-lessrecombinase gene in a plasmid vector. This construct is introduced intothe target organism which carries an antibiotic resistance gene flankedby resolvase sites. Growth in the presence of the antibiotic removesfrom the population those fragments cloned into the plasmid vectorcapable of supporting transcription of the recombinase gene andtherefore have caused loss of antibiotic resistance. The resistant poolis introduced into a host and at various times after infection bacteriamay be recovered and assessed for the presence of antibiotic resistance.The chromosomal fragment carried by each antibiotic sensitive bacteriumshould carry a promoter or portion of a gene normally upregulated duringinfection. Sequencing upstream of the recombinase gene allowsidentification of the up regulated gene.

RT-PCR may also be used to analyze gene expression patterns. For RT PCRusing the polynucleotides of the invention, messenger RNA is isolatedfrom bacterial infected tissue, e.g., 48 hour murine lung infections,and the amount of each mRNA species assessed by reverse transcription ofthe RNA sample primed with random hexanucleotides followed by PCR withgene specific primer pairs. The determination of the presence and amountof a particular mRNA species by quantification of the resultant PCRproduct provides information on the bacterial genes which aretranscribed in the infected tissue. Analysis of gene transcription canbe carried out at different times of infection to gain a detailedknowledge of gene regulation in bacterial pathogenesis allowing for aclearer understanding of which gene products represent targets forscreens for antibacterials. Because of the gene specific nature of thePCR primers employed it should be understood that the bacterial mRNApreparation need not be free of mammalian RNA. This allows theinvestigator to carry out a simple and quick RNA preparation frominfected tissue to obtain bacterial mRNA species which are very shortlived in the bacterium (in the order of 2 minute halflives). Optimallythe bacterial mRNA is prepared from infected murine lung tissue bymechanical disruption in the presence of TRIzole (GIBCO-BRL) for veryshort periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of Streptococcus pneumoniae 16Sribosomal RNA as detected by probing Northerns with a suitably labeledsequence specific oligonucleotide probe. Typically, a 5′ dye labeledprimer is used in each PCR primer pair in a PCR reaction which isterminated optimally between 8 and 25 cycles. The PCR products areseparated on 6% polyacrylamide gels with detection and quantificationusing GeneScanner (manufactured by ABI).

Gridding and Polynucleotide Subtraction

Methods have been described for obtaining information about geneexpression and identity using so called “high density DNA arrays” orgrids. See, e.g., M. Chee et al., Science, 274:610-614 (1996) and otherreferences cited therein. Such gridding assays have been employed toidentify certain novel gene sequences, referred to as Expressed SequenceTags (EST) (Adams et a., Science, 252:1651-1656 (1991)). A variety oftechniques have also been described for identifying particular genesequences on the basis of their gene products. For example, seeInternational Patent Application No. WO91/07087, published May 30, 1991.In addition, methods have been described for the amplification ofdesired sequences. For example, see International Patent Application No.WO01/17271, published Nov. 14, 1991.

The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probes obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularlyStreptococcus pneumoniae, and may be useful in diagnosing and/orprognosing disease or a course of disease. A grid comprising a number ofvariants of the polynucleotide sequence of SEQ ID NO:1 are preferred.Also preferred is a comprising a number of variants of a polynucleotidesequence encoding the polypeptide sequence of SEQ ID NO:2.

Antibodies

The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively.

In certain preferred embodiments of the invention there are providedantibodies against fabZ polypeptides or polynucleotides.

Antibodies generated against the polypeptides or polynucleotides of theinvention can be obtained by administering the polypeptides and/orpolynucleotides of the invention, or epitope-bearing fragments of eitheror both, analogues of either or both, or cells expressing either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artprovides antibodies produced by continuous cell line cultures can beused. Examples include various techniques, such as those in Kohler, G.and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., ImmunologyToday 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES ANDCANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-fabZ or from naivelibraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks, etal., (1992) Biotechnology 10, 779-783). The affinity of these antibodiescan also be improved by, for example, chain shuffling (Clackson et al.,(1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides or polynucleotides of the inventionto purify the polypeptides or polynucleotides by, for example, affinitychromatography.

Thus, among others, antibodies against fabZ-polypeptide orfabZ-polynucleotide may be employed to treat infections, particularlybacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

A polypeptide or polynucleotide of the invention, such as anantigenically or immunologically equivalent derivative or a fusionprotein of the polypeptide is used as an antigen to immunize a mouse orother animal such as a rat or chicken. The fusion protein may providestability to the polypeptide. The antigen may be associated, for exampleby conjugation, with an immunogenic carrier protein for example bovineserum albumin, keyhole limpet haemocyanin or tetanus toxoid.Alternatively, a multiple antigenic polypeptide comprising multiplecopies of the polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized,” where thecomplimentarity determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

In accordance with an aspect of the invention, there is provided the useof a polynucleotide of the invention for therapeutic or prophylacticpurposes, in particular genetic immunization. Among the particularlypreferred embodiments of the invention are naturally occurring allelicvariants of fabZ polynucleotides and polypeptides encoded thereby.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet(1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419),delivery of DNA complexed with specific protein carriers (Wu et al., J.Biol Chem. (1989) 264: 16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science (1989) 243: 375), particle bombardment (Tang et al., Nature(1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and invivo infection using cloned retroviral vectors (Seeger et al., PNAS USA(1984) 81:5849).

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases hereinbefore mentioned. It istherefore desirable to devise screening methods to identify compoundswhich stimulate or which inhibit the function of the polypeptide orpolynucleotide. Accordingly, in a further aspect, the present inventionprovides for a method of screening compounds to identify those whichstimulate or which inhibit the function of a polypeptide orpolynucleotide of the invention, as well as related polypeptides andpolynucleotides. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of fabZ polypeptides and polynucleotides; ormay be structural or functional mimetics thereof (see Coligan et al.,Current Protocols in Immunology 1 (2): Chapter 5 (1991)).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide or polynucleotide, as the case may be.Further, the screening methods may simply comprise the steps of mixing acandidate compound with a solution containing a polypeptide orpolynucleotide of the present invention, to form a mixture, measuringfabZ polypeptide and/or polynucleotide activity in the mixture, andcomparing the fabZ polypeptide and/or polynucleotide activity of themixture to a standard. Fusion proteins, such as those made from Fcportion and fabZ polypeptide, as hereinbefore described, can also beused for high-throughput screening assays to identify antagonists of thepolypeptide of the present invention, as well as phylogenetically andand/or functionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentswhich may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) he action of fabZpolypeptides or polynucleotides, particularly those compounds that arebacteristatic and/or bactericidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising fabZ polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a fabZ agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the fabZ polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of fabZ polypeptide aremost likely to be good antagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in fabZ polynucleotide or polypeptideactivity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance,¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide which compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by fabZ polypeptide associating with anotherfabZ polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of fabZ polypeptide dimers,trimers, tetramers or higher order structures, or structures formed byfabZ polypeptide bound to another polypeptide. FabZ polypeptide can belabeled with both a donor and acceptor fluorophore. Upon mixing of thetwo labeled species and excitation of the donor fluorophore. Upon mixingof the two labeled species and excitation of the donor fluorophore,fluorescence energy transfer can be detected by observing fluorescenceof the acceptor. Compounds that block dimerization will inhibitfluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on fabZ polypeptide self-association as well as an associationof fabZ polypeptide and another polypeptide or small molecule. fabZpolypeptide can be coupled to a sensor chip at low site density suchthat covalently bound molecules will be monomeric. Solution protein canthen passed over the fabZ polypeptide-coated surface and specificbinding can be detected in real-time by monitoring the change inresonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for fabZ polypeptideself-association as well as an association of fabZ polypeptide andanother polypeptide or small molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of fabZ polypeptide with another fabZpolypeptide or a different polypeptide. fabZ polypeptide can be coupledto a scintillation-filled bead. Addition of radio-labeled fabZpolypeptide results in binding where the radioactive source molecule isin close proximity to the scintillation fluid. Thus, signal is emittedupon fabZ polypeptide binding and compounds that prevent fabZpolypeptide self-association or an association of fabZ polypeptide andanother polypeptide or small molecule will diminish signal.

ICS biosensors have been described by AMBRI (Australian MembraneBiotechnology Research Institute). They couple the self-association ofmacromolecules to the closing of gramacidin-facilitated ion channels insuspended membrane bilayers and hence to a measurable change in theadmittance (similar to impedance) of the biosensor. This approach islinear over six decades of admittance change and is ideally suited forlarge scale, high through-put screening of small molecule combinatoriallibraries.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for fabZ agonists is a competitive assaythat combines fabZ and a potential agonist with fabZ-binding molecules,recombinant fabZ binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. FabZ can be labeled, such as radioactivityor a colorimetric compound, such that the number of fabZ molecules boundto a binding molecule or converted to product can be determinedaccurately to assess the effectiveness of the potential antagonist.

Potential antagonists include, among others, small organic molecules,peptides, polypeptides and antibodies that bind to a polynucleotideand/or polypeptide of the invention and thereby inhibit or extinguishits activity or expression. Potential antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds the same sites on a binding molecule,such as a binding molecule, without inducing fabZ-induced activities,hereby preventing the action or expression of fabZ polypeptides and/orpolynucleotides by excluding fabZ polypeptides and/or polynucleotidesfrom binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Koano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of fabZ.

Other examples of potential polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

Certain of the polypeptides of the invention are biomimetics, functionalmimetics of the natural fabZ polypeptide. These functional mimetics maybe used for, among other things, antagonizing the activity of fabZpolypeptide or as a antigen or immunogen in a manner described elsewhereherein. Functional mimetics of the polypeptides of the invention includebut are not limited to truncated polypeptides. For example, preferredfunctional mimetics include, a polypeptide comprising the polypeptidesequence set forth in SEQ ID NO:2 lacking 20, 30, 40, 50, 60, 70 or 80amino- or carboxy-terminal amino acid residues, including fusionproteins comprising one or more of these truncated sequences.Polynucleotides encoding each of these functional mimetics may be usedas expression cassettes to express each mimetic polypeptide. It ispreferred that these cassettes comprise 5′ and 3′ restriction sites toallow for a convenient means to ligate the cassettes together whendesired. It is further preferred that these cassettes comprise geneexpression signals known in the art or described elsewhere herein.

Thus, in another aspect, the present invention relates to a screeningkit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for a polypeptide and/or polynucleotide of thepresent invention; or compounds which decrease or enhance the productionof such polypeptides and/or polynucleotides, which comprises: (a) apolypeptide and/or a polynucleotide of the present invention; (b) arecombinant cell expressing a polypeptide and/or polynucleotide of thepresent invention; (c) a cell membrane expressing a polypeptide and/orpolynucleotide of the present invention; (d) antibody to a polypeptideand/or polynucleotide of the present invention; which polypeptide ispreferably that of SEQ ID NO:2, and which polynucleotide is preferablythat of SEQ ID NO:1.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist, antagonist orinhibitor of the polypeptide and/or polynucleotide, by: (a) determiningin the first instance the three-dimensional structure of the polypeptideand/or polynucleotide, or complexes thereof; (b) deducing thethree-dimensional structure for the likely reactive site(s), bindingsite(s) or motif(s) of an agonist, antagonist or inhibitor; (c)synthesizing candidate compounds that are predicted to bind to or reactwith the deduced binding site(s), reactive site(s), and/or motif(s); and(d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

It will be further appreciated that this will normally be an iterativeprocess, and this iterative process may be performed using automated andcomputer-controlled steps.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of fabZ polypeptide and/or polynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the fabZpolypeptide and/or polypeptide.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

In still another approach, expression of the gene encoding endogenousfabZ polypeptide can be inhibited using expression blocking techniques.This blocking may be targeted against any step in gene expression, butis preferably targeted against transcription and/or translation. Anexamples of a known technique of this sort involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J. Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered pe se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial fabZ proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

In accordance with yet another aspect of the invention, there areprovided fabZ agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein “H. pylori”) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyof Research on Cancer, Lyon, France, http://ww.uicc.ch/ecp/ecp2904.htm).Moreover, the International Agency for Research on Cancer recentlyrecognized a cause-and-effect relationship between H. pylori and gastricadenocarcinoma, classifying the bacterium as a Group I (definite)carcinogen. Preferred antimicrobial compounds of the invention (agonistsand antagonists of fabZ polypeptides and/or polynucleotides) found usingscreens provided by the invention, or known in the art, particularlynarrow-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also prevent, inhibit and/or cure gastric ulcers andgastritis.

Vaccines

There are provided by the invention, products, compositions and methodsfor assessing fabZ expression, treating disease, assaying geneticvariation, and administering a fabZ polypeptide and/or polynucleotide toan organism to raise an immunological response against a bacteria,especially a Streptococcus pneumoniae bacteria.

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with fabZ polynucleotide and/orpolypeptide, or a fragment or variant thereof, adequate to produceantibody and/or T cell immune response to protect said individual frominfection, particularly bacterial infection and most particularlyStreptococcus pneumoniae infection. Also provided are methods wherebysuch immunological response slows bacterial replication. Yet anotheraspect of the invention relates to a method of inducing immunologicalresponse in an individual which comprises delivering to such individuala nucleic acid vector, sequence or ribozyme to direct expression of fabZpolynucleotide and/or polypeptide, or a fragment or a variant thereof,for expressing fabZ polynucleotide and/or polypeptide, or a fragment ora variant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual, preferably a human, from disease, whether that diseaseis already established within the individual or not. One example ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, aDNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a fabZ polynucleotideand/or polypeptide encoded therefrom, wherein the composition comprisesa recombinant fabZ polynucleotide and/or polypeptide encoded therefromand/or comprises DNA and/or RNA which encodes and expresses an antigenof said fabZ polynucleotide, polypeptide encoded therefrom, or otherpolypeptide of the invention. The immunological response may be usedtherapeutically or prophylactically and may take the form of antibodyimmunity and/or cellular immunity, such as cellular immunity arisingfrom CTL or CD4+ T cells.

A fabZ polypeptide or a fragment thereof may be fused with co-protein orchemical moiety which may or may not be itself produce antibodies, butwhich is capable of stabilizing the first protein and producing a fusedor modified protein which will have antigenic and/or immunogenicproperties, and preferably protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, or any other relatively large co-proteinwhich solubilizes the protein and facilitates production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system ofthe organism receiving the protein. The co-protein may be attached toeither the amino- or carboxy-terminus of the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Streptococcus pneumoniae. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Streptococcus pneumoniae infection, inmammals, particularly humans.

A polypeptide of the invention may be used as an antigen for vaccinationof a host to produce specific antibodies which protect against invasionof bacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, for example, by mechanical, chemical, thermal orradiation damage or by implantation of indwelling devices, or wounds inthe mucous membranes, such as the mouth, throat, mammary glands, urethraor vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual,and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain fabZpolypeptides and polynucleotides, it is to be understood that thiscovers fragments of the naturally occurring polypeptides andpolynucleotides, and similar polypeptides and polynucleotides withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant polypeptides orpolynucleotides.

Compositions, Kits and Administration

In a further aspect of the invention there are provided compositionscomprising a fabZ polynucleotide and/or a fabZ polypeptide foradministration to a cell or to a multicellular organism.

The invention also relates to compositions comprising a polynucleotideand/or a polypeptides discussed herein or their agonists or antagonists.The polypeptides and polynucleotides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to an individual. Such compositionscomprise, for instance, a media additive or a therapeutically effectiveamount of a polypeptide and/or polynucleotide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration. The invention further relates todiagnostic and pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of the invention maybe employed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide and/or polynucleotide, such as the soluble form of apolypeptide and/or polynucleotide of the present invention, agonist orantagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds,

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred form of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible, Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, and continuous ambulatory peritoneal dialysis (CAPD)catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden preoperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable informationresource with which to determine their 2- and 3-dimensional structuresas well as to identify further sequences of similar homology. Theseapproaches are most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as GCC.

The polynucleotide and polypeptide sequences of the invention areparticularly useful as components in databases useful for searchanalyses as well as in sequence analysis algorithms. As used in thissection entitled “Sequence Databases, Sequences in a Tangible Medium,and Algorithms,” and in claims related to this section, the terms“polynucleotide of the invention” and “polynucleotide sequence of theinvention” mean any detectable chemical or physical characteristic of apolynucleotide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form. For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, called bases, and mass spectrographic data, As used in thissection entitled Databases and Algorithms and in claims related thereto,the terms “polypeptide of the invention” and “polypeptide sequence ofthe invention” mean any detectable chemical or physical characteristicof a polypeptide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form, For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, and mass spectrographic data.

The invention provides a computer readable medium having stored thereonpolypeptide sequences of tho invention and/or polynucleotide sequencesof the invention. For example, a computer readable medium is providedcomprising and having stored thereon a member selected from the groupconsisting of: a polynucleotide comprising the sequence of apolynucleotide of the invention; a polypeptide comprising the sequenceof a polypeptide sequence of the invention; a set of polynucleotidesequences wherein at least one of the sequences comprises the sequenceof a polynucleotide sequence of the invention; a set of polypeptidesequences wherein at least one of the sequences comprises the sequenceof a polypeptide sequence of the invention; a data set representing apolynucleotide sequence comprising the sequence of polynucleotidesequence of the invention, a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of apolypeptide sequence of the invention; a polynucleotide comprising thesequence of a polynucleotide sequence of the invention; a polypeptidecomprising the sequence of a polypeptide sequence of the invention; aset of polynucleotide sequences wherein at least one of the sequencescomprises the sequence of a polynucleotide sequence of the invention; aset of polypeptide sequences wherein at least one of said sequencescomprises the sequence of a polypeptide sequence of the invention; adata set representing a polynucleotide sequence comprising the sequenceof a polynucleotide sequence of the invention; a data set representing apolynucleotide sequence encoding a polypeptide sequence comprising thesequence of a polypeptide sequence of the invention. The computerreadable medium can be any composition of matter used to storeinformation or data, including, for example, commercially availablefloppy disks, tapes, chips, hard drives, compact disks, and video disks.

Also provided by the invention are methods for the analysis of charactersequences or strings, particularly genetic sequences or encoded geneticsequences, Preferred methods of sequence analysis include, for example,methods of sequence homology analysis, such as identity and similarityanalysis, RNA structure analysis, sequence assembly, cladistic analysis,sequence motif analysis, open reading frame determination, nucleic acidbase calling, nucleic acid base trimming, and sequencing chromatogrampeak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of providing a firstpolynucleotide sequence comprising the sequence a polynucleotide of theinvention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

A computer based method is also provided for performing homologyidentification, said method comprising the steps of, providing a firstpolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said firstpolypeptide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

A computer based method is still further provided for polynucleotideassembly, said method comprising the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and atleast one second polynucleotide or polypeptide sequence,

A computer based method is still further provided for polynucleotideassembly, said method comprising the steps of providing a firstpolypeptide sequence comprising a polypeptide of the invention in acomputer readable medium; and screening for at least one overlappingregion between said first polypeptide sequence and at least one secondpolynucleotide or polypeptide sequence.

Preferred embodiments of the assemble methods of the invention use theassembly method set forth in U.S. Pat. No. 5,618,672.

In another preferred embodiment of the invention there is provided acomputer readable medium having stored thereon a member selected fromthe group consisting of a polynucleotide comprising the sequence of SEQID NO:1; a polypeptide comprising the sequence of SEQ ID NO:2; a set ofpolynucleotide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO:1; a set of polypeptide sequenceswherein at least one of said sequences comprises the sequence of SEQ IDNO:2; a data set representing a polynucleotide sequence comprising thesequence of SEQ ID NO:1; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of SEQID NO:2; a polynucleotide comprising the sequence of SEQ ID NO:1; apolypeptide comprising the sequence of SEQ ID NO:2; a set ofpolynucleotide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO:1; a set of polypeptide sequenceswherein at least one of said sequences comprises the sequence of SEQ IDNO:2; a data set representing a polynucleotide sequence comprising thesequence of SEQ ID NO:1; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of SEQID NO:2. A further preferred embodiment of the invention provides acomputer based method for performing homology identification, saidmethod comprising the steps of providing a polynucleotide sequencecomprising the sequence of SEQ ID NO:1 in a computer readable medium;and comparing said polynucleotide sequence to at least onepolynucleotide or polypeptide sequence to identify homology.

A still further preferred embodiment of the invention provides acomputer based method for performing homology identification, saidmethod comprising the steps of: providing a polypeptide sequencecomprising the sequence of SEQ ID NO:2 in a computer readable medium;and comparing said polypeptide sequence to at least one polynucleotideor polypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps ofproviding a first polynucleotide sequence comprising the sequence of SEQID NO:1 in a computer readable medium; and screening for at least oneoverlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polynucleotide sequence comprising the sequence of SEQID NO:1 in a computer readable medium; and comparing said polynucleotidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Antibody(ies)” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Antigenically equivalent derivative(s)” as used herein encompasses apolypeptide, polynucleotide, or the equivalent of either which will bespecifically recognized by certain antibodies which, when raised to theprotein, polypeptide or polynucleotide according to the invention,interferes with the immediate physical interaction between pathogen andmammalian host.

“Bispecific antibody(ies)” means an antibody comprising at least twoantigen binding domains, each domain directed against a differentepitope.

“Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

“Fusion protein(s)” refers to a protein encoded by two, often unrelated,fused genes or fragments thereof. In one example, EP-A-0464 disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

“Host cell(s)” is a cell which has been transformed or transfected, oris capable transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al, J. Mol. Biol. 215, 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA, 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons,

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n_(n)≦x_(n)−(x_(n)·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby after the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:1, that is itmay be 100% identical, or it may include up to a certain integer numberof nucleic acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of nucleic acids in SEQ ID NO:1 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of nucleic acids in SEQ ID NO:1, or:

n_(n)≦x_(n)−(x_(n)·y),

wherein n_(n) is the number of nucleic acid alterations, x_(n) is thetotal number of nucleic acids in SEQ NO:1, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO:2 or may include up to a certain integer number ofamino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n_(a)≦x_(z)−(x_(a)·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n_(a)≦x_(a)−(x_(a)·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

“Immunologically equivalent derivative(s)” as used herein encompasses apolypeptide, polynucleotide, or the equivalent of either which when usedin a suitable formulation to raise antibodies in a vertebrate, theantibodies act to interfere with the immediate physical interactionbetween pathogen and mammalian host.

“Immunospecific” means that characteristic of an antibody whereby itpossesses substantially greater affinity for the polypeptides of theinvention or the polynucleotides of the invention than its affinity forother related polypeptides or polynucleotides respectively, particularlythose polypeptides and polynucleotides in the prior art.

“Individual(s)” means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e:, if it occurs in nature, has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide orpolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any-otherrecombinant method is “isolated” even if it is a present in saidorganism, which organism may be living or non-living.

“Organism(s)” means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinohacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neissea gonorrheae, Neisseria meningitis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfingens, Clostridiumtetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsiiand Chlamydia trachomitis, (ii) an archaeon, including but not limitedto Archaebacter, and (iii) a unicellular or filamentous eukaryote,including but not limited to, a protozoan, a fungus, a member of thegenus Saccharomyces, Kluveromyces, or Candida, and a member of thespecies Saccharomyces ceriviseae, Kluveromyces lactis, or Candidaalbicans.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single-and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells,“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins, Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or vary degree at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications. Modificationscan occur anywhere in a polypeptide, including the peptide backbone, theamino acid side-chains, and the amino or carboxyl termini. Modificationsinclude, for example, acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wolf, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Subtraction set” is one or more, but preferably less than 100,polynucleotides comprising at least one polynucleotide of the invention

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide, Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr,Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain Selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO:1. Librariesmay be prepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods,

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PaII, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 fabZ Characterization

There are four distinct chemical reactions required to complete eachround of fatty acid elongation in Escherichia coil, which is theparadigm for dissociated (type II) fatty acid synthase system. The thirdstep of this pathway is the dehydration of the nascent β-hydroxyacyl-ACPto trans-2-acyl-ACP and this step is catalyzed by FabZ. Specifically,FabZ dehydratase catalyzes the dehydration of short chain and long chainsaturated and unsaturated β-hydroxyacyl-ACP. It does not have isomeraseactivity therefore it is not involved in the biosynthesis of unsaturatedfatty acids. FabZ has been genetically and enzymatically characterizedin E. coli which can be found in the following references: Richard J.Heath, Charles O. Rock, Roles of FabA and FabZ b-hydroxyacyl-AcylCarrier Protein Dehydratases in Escherichia coli Fatty acidBiosynthesis, Journal of Biological Chemistry (1996), 271, 27795-27801;and Mohan, S., Kelly, T. M., Eveland, S. S., Raetz, C. R. H., andAnderson, M. S., (1994) J Biol. Chem. (1994) 269, 32896-32903.

2 1 423 DNA Streptococcus pneumoniae CDS (1)...(420) 1 atg atc gat attcaa gga atc aaa gaa gct ctt ccc cac cgt tat cct 48 Met Ile Asp Ile GlnGly Ile Lys Glu Ala Leu Pro His Arg Tyr Pro 1 5 10 15 atg ctt cta gtggac cgt gtc ttg gaa gtg agc gag gat acc att gtt 96 Met Leu Leu Val AspArg Val Leu Glu Val Ser Glu Asp Thr Ile Val 20 25 30 gct atc aaa aat gtgacc atc aac gag cct ttc ttt aac ggc cac ttt 144 Ala Ile Lys Asn Val ThrIle Asn Glu Pro Phe Phe Asn Gly His Phe 35 40 45 cct caa tac cca gtt atgcca ggt gtt gtg att atg gaa gcc ttg gcg 192 Pro Gln Tyr Pro Val Met ProGly Val Val Ile Met Glu Ala Leu Ala 50 55 60 caa act gcc ggt gtg ttg gagtta tca aaa cct gaa aat aaa gga aaa 240 Gln Thr Ala Gly Val Leu Glu LeuSer Lys Pro Glu Asn Lys Gly Lys 65 70 75 80 ctg gtc ttt tac gct ggt atggac aag gtt aag ttc aag aag caa gtt 288 Leu Val Phe Tyr Ala Gly Met AspLys Val Lys Phe Lys Lys Gln Val 85 90 95 gta cca ggc gac caa ttg gtt atgaca gcg act ttt gta aaa cgt cgt 336 Val Pro Gly Asp Gln Leu Val Met ThrAla Thr Phe Val Lys Arg Arg 100 105 110 ggc acc ata gct gtg gtt gaa gcaaag gct gaa gtg gat ggc aag ctt 384 Gly Thr Ile Ala Val Val Glu Ala LysAla Glu Val Asp Gly Lys Leu 115 120 125 gca gcc agt ggt acc ctt act tttgca att ggg aac taa 423 Ala Ala Ser Gly Thr Leu Thr Phe Ala Ile Gly Asn130 135 140 2 140 PRT Streptococcus pneumoniae 2 Met Ile Asp Ile Gln GlyIle Lys Glu Ala Leu Pro His Arg Tyr Pro 1 5 10 15 Met Leu Leu Val AspArg Val Leu Glu Val Ser Glu Asp Thr Ile Val 20 25 30 Ala Ile Lys Asn ValThr Ile Asn Glu Pro Phe Phe Asn Gly His Phe 35 40 45 Pro Gln Tyr Pro ValMet Pro Gly Val Val Ile Met Glu Ala Leu Ala 50 55 60 Gln Thr Ala Gly ValLeu Glu Leu Ser Lys Pro Glu Asn Lys Gly Lys 65 70 75 80 Leu Val Phe TyrAla Gly Met Asp Lys Val Lys Phe Lys Lys Gln Val 85 90 95 Val Pro Gly AspGln Leu Val Met Thr Ala Thr Phe Val Lys Arg Arg 100 105 110 Gly Thr IleAla Val Val Glu Ala Lys Ala Glu Val Asp Gly Lys Leu 115 120 125 Ala AlaSer Gly Thr Leu Thr Phe Ala Ile Gly Asn 130 135 140

What is claimed is:
 1. An isolated polynucleotide segment comprising anucleic acid sequence which encodes a polypeptide consisting of SEQ IDNO:2, wherein the nucleic acid sequence is not genomic DNA.
 2. A vectorcomprising the isolated polynucleotide segment of claim
 1. 3. Anisolated host cell comprising the vector of claim
 2. 4. A process forproducing a polypeptide comprising culturing the host cell of claim 3,under conditions sufficient for the production of the polypeptide,wherein the polypeptide consists of SEQ ID NO:2.
 5. A vector with aninsert comprising a polynucleotide sequence which encodes a polypeptideconsisting of SEQ ID NO:2 with a heterologous amino acid sequence fusedthereto.
 6. A process for producing a polypeptide comprising culturingthe host cell of claim 5 to produce the polypeptide, wherein thepolypeptide consists of a heterologous amino acid sequence fused to SEQID NO:2.
 7. A vector with an insert comprising a polynucleotide sequencewherein the polynucleotide sequence encodes a polypeptide comprising theamino acid sequence set forth in SEQ ID NO:2 and wherein thepolynucleotide sequence comprises a heterologous promoter positioned topromote expression of a mRNA for which transcription of the open readingframe initiates at amino acid 1 of SEQ ID NO:2.
 8. An isolated host cellcomprising the vector of claim
 7. 9. A process for producing thepolypeptide comprising culturing the host cell of claim 8 underconditions sufficient for the production of the polypeptide.